KR20140123553A - Immunogenic Composition - Google Patents

Abstract

The invention yen. Methoxy Ninja ET display (N. meningitidis) to provide immunogenic polysaccharide protein-polymer fabric and a method comprising the capsular polysaccharide from serogroup X. The present invention is a conjugated yen prepared by the reaction. ≪ RTI ID = 0.0 > Meninitidis X polysaccharide-carrier protein < / RTI > Thus, the present invention relates to a polyacrylamide polysaccharide protein conjugate composition comprising a capsular saccharide from serogroup X and one or more encapsulated polysaccharides from serogroups A, C, W135 and Y, wherein i) the polysaccharide A, C, W135 and X are mechanically sized while the polysaccharide Y is chemically sized, ii) all saccharides are incorporated into the carrier protein via a linker by cyanocatalytic chemistry, and iii) , And iv) two or more different carrier proteins selected from the group consisting of TT, DT and CRM197.

Description

Immunogenic composition

Nyseria Eighty-mail Ninja display (Neisseria meningitidis (meningococcus) is a gram negative human pathogen. It forms colony in the pharynx causing meningitis and occasionally sepsis without meningitis. Although this is one feature that clearly distinguish between the presence of the meningococcal polysaccharide capsule present in all pathogenic meningococcal, yen is closely linked to and in Kono In (N. gonorrhoeae). Based on the capsular polysaccharide of the organism yen. Methoxy Ninja ET display 12 more serogroup was confirmed in (N. meningitidis) (A, B , C, H, I, K, L, 29E, W135, X, Y and Z).

Serum A ('MenA') is the most common cause of contagious disease in sub-Saharan Africa. Serum B and C are responsible for most cases in developed countries and the rest are caused by serogroups W135 and Y.

Vaccines using only polysaccharides (PS) have relatively low immunogenicity. To address the relatively low immunogenicity of polysaccharides, conjugating PS vaccines to protein carriers increases immunogenicity and provides long-term protection in children. Numerous meningococcal conjugate vaccines have been approved and are being sold worldwide. " Nysealia Examples of methoxy Ninja ET discharge port copolymer "known such vaccines as is monovalent meningococcal A PO copolymer (MenAfriVac), monovalent meningococcal C PO copolymer (Meningitec) and tetravalent ACYW meningococcal PO copolymer (Menveo and Menactra) to be.

Capsule polysaccharides have been used for vaccination as well as for classification. Injection of capsular polysaccharides from serogroups A, C, Y, and W135. The tetravalent vaccine has been known for many years and has been approved for human use. Although effective for adolescents and adults, it causes insufficient immune responses and short term protection and can not be used in infants. Both Mencevax ACWY ™ and Menomune ™ contain 50 μg of purified polysaccharide, respectively, once reconstituted from their lyophilized form. The capsular saccharides of serogroups A, C, W135 and Y are also formulated in the form of a complex to provide a quadrivalent vaccine, for example, a special Menactra ™ product. In addition, the incorporated serogroup A polysaccharide was approved for human use as MenAfriVac ™, while serogroup C oligosaccharides were approved for human use as Menjugate ™, Meningitec ™ and NeisVac-C ™.

yen. Meninitidis serogroup X strains were first introduced in the 1960s and isolated in several cases of invasive meningococcal disease in North America, Europe, Australia and China. yen. The occurrence of itty-mail Ninja display serogroup X strains were reported in Niger, Kenya, western and northern Ghana. yen. Meninitidis serogroup X strains have been reported to be highly effective for colony formation among British recruits (Abdullah Kilic et al .; Neisseria meningitidis Serogroup X Sequence Type 767 in Turkey; Journal of Clinical Microbiology, Nov. 2010, p. 4340-4341; Vol. 48, No. 11). Repeated massive vaccinations in many African countries have been reported to have contributed to colonization by meningococcal disease due to serogroup X strains and may have resulted in a changed profile of meningococcal disease (Gagneux, S. P et al., Prospective study of a serogroup X Neisseria meningitidis outbreak in northern Ghana, J. Infect. Dis. 185: 618-626;

Serum B, C, Y and W135 capsular polysaccharides of meningococci are composed of sialic acid derivatives. Serogroup B and C meningococci express (a2-8) - and (a2-9 239) -conjugated polycialic acid, respectively, while alternating sequences of D-glucose or D- galactoside and sialic acid are expressed in serogroup Y and W135 yen. It is expressed by meninitidis . In contrast, the serogroup A meningococcal capsule is (α1-6) - N- acetyl mannose amine 6-phosphate, whereas the associated yen. Methoxy Ninja ET discharge serogroup X is (α1-4) - synthesizing an N- acetyl-linked glucose 1-phosphate capsule of the amine-type polymer (lit. [Yih-Ling Tzeng et al .; Genetic Basis for Biosynthesis of the (134) - Linked N-Acetyl-D-Glucosamine 1-Phosphate Capsule of Neisseria meningitidis Serogroup X; Infection And Immunity, Dec. 2003, p. 6712-6720; Vol. 71, No. 12).

The existing meningococcal conjugate vaccine is based on the A C Y W135 polysaccharide. The increased incidence of MenX disease in the African meningitis belt over the past five years [1,4] assures the development and introduction of the MenX polysaccharide conjugate vaccine in selected regions to prevent and control future infectious diseases.

Despite the availability of comprehensive serum prevalence and structural data for Meningococci X, commercially viable synthetic vaccines, including X polysaccharides, have been shown to have extremely limited success with respect to their purification, incorporation and formulation stability aspects Were reported to have not yet been developed. This is particularly Neisseria Neisseria containing Meninitidis X polysaccharide It provides an additional challenge to successfully handle and control various parameters when applying a scalable incorporation process for large-scale manufacture of the meninitidis formulations.

The present invention has been made from the surprising discovery that it is possible to produce monovalent or multivalent immunogenic compositions based on the meningococcal polysaccharide formulations from serogroup X using an expandable and efficient incorporation process.

I) polysaccharide sizing, ii) CPPT-based activation of a sized polysaccharide having an average molecular weight between 100 kDa and 150 kDa at a pH between 9 and 9.5, iii) a period of about 2 to 5 minutes ADH addition followed by incubation for 4 to 20 hours iv) reaction of the ADH-activated polysaccharide with the purified inactivated carrier protein at a ratio between 0.75 and 1.5 in the presence of MES buffer and EDAC followed by incubation for 3 to 4 hours at constant temperature The yen produced by the inclusion reaction, including treatment . The present invention relates to a synergistically effective amount of a saccharide- mannitolite X-sugar-carrier protein conjugate, wherein the incorporation reaction is carried out at 2 ° C to 8 ° C, the yield of the formulator is 20% to 30% Is from 0.2 to about 0.6.

Alternatively, the yen. Methoxy Ninja ET discharge X saccharide-carrier protein PO copolymers are also i) polysaccharides, sizing, ii) 9 and of the sizing polysaccharide at a pH between 9.5, having an average molecular weight between 100 kDa and 150 kDa CPPT based activation, iii) 2 bun Followed by the addition of an ADH-activated carrier protein at a ratio of saccharide to protein between 0.5 and 2 after 3 minutes, followed by incubation for 2 to 20 hours, wherein the incorporation reaction is 22 Deg.] C to about 25 < 0 > C, and the yield of the formulations is from 5% to about 10%.

Thus, the present invention relates to a polyacrylamide polysaccharide protein conjugate composition comprising a capsular saccharide from serogroup X and one or more encapsulated polysaccharides from serogroups A, C, W135 and Y, wherein i) the polysaccharide A, C, W135 and X are mechanically sized, while polysaccharide Y is chemically sized, ii) all saccharides are incorporated into the carrier protein via a linker by cyanogen synthesis chemistry, and iii) all of the proteins in the final construct The ratio of saccharides is between 0.2 and 0.6, and iv) two or more different carrier proteins selected from the group consisting of TT, DT and CRM197.

Figure 1 shows an overlay of inclusion reactions when Men X Ps (215 kDa) is incorporated into the hydrazine derivatized TT.
Figure 2 shows the purified form when Men X Ps (215 kDa) is incorporated into the hydrazine derivatized TT.
Figure 3 shows an overlay of the inclusion response when Men X Ps (326 kDa) is incorporated into the ADH derivatized TT.
Figure 4 shows the purified form of Men X Ps (326 kDa) when incorporated into ADH derivatized TT.
Figure 5 shows an overlay of the inclusion response when Men X Ps (120 kDa) is incorporated into the ADH derivatized TT.
Figure 6 shows the purified form of Men X Ps (120 kDa) when incorporated into ADH derivatized TT.
Figure 7 shows the chromatogram of the meningococcal X polysaccharide originally.
Figure 8 shows an overlay of the inclusion response when Men X Ps (510 kDa) is activated with ADH and incorporated into the purified TT.
Figure 9 shows a purified conjugate when Men X Ps (510 kDa) is activated with ADH and incorporated into purified TT.
Figure 10 shows an overlay of the inclusion response when Men X Ps (250 kDa) is activated with ADH and incorporated into the purified TT.
Figure 11 shows a purified form of Men X Ps (250 kDa) to be incorporated into purified TT activated with ADH.

"Multi-immunogenic composition" means the following.

Composition < RTI ID = 0.0 > I < / RTI & gt ; PO copolymers, (b) a serogroup C yen methoxy ninja ET discharge capsular saccharides (i) and tetanus toxoid (ii). A mixture of capsular saccharide (i) and tetanus toxoid (ii) of meninitidis, (c) serum group Y yen. PO copolymers, (d) serogroup W135 of yen methoxy ninja ET discharge capsular saccharides (i) and diphtheria toxoid (ii). PO copolymers, and (e) of serogroup X yen methoxy ninja ET discharge capsular saccharides (i) and tetanus toxoid (ii). And a complex of a capsular saccharide (i) and a tetanus toxoid (ii) of meninitidis.

Composition II comprises (a) Serum Group A ; PO copolymers, (b) a serogroup C yen methoxy ninja ET discharge capsular saccharides (i) and tetanus toxoid (ii). Four polymers of methoxy Ninja ET discharge of the capsule-type saccharides (i) and CRM197 (ii), (c) serogroup Y yen. PO copolymers, (d) serogroup W135 of yen methoxy ninja ET discharge capsular saccharides (i) and tetanus toxoid (ii). PO copolymers, and (e) of serogroup X yen methoxy Ninja ET discharge of the capsule-type saccharides (i) and CRM197 (ii). Methoxy and a capsule of the capsule-polymer sugars (i) ET D ninja's and CRM197 (ii).

Composition III is composed of (a) serotypes A and B; PO copolymers, (b) a serogroup C yen methoxy Ninja ET discharge of the capsule-type saccharides (i) and CRM197 (ii). Four polymers of methoxy Ninja ET discharge of the capsule-type saccharides (i) and CRM197 (ii), (c) serogroup Y yen. PO copolymers, (d) serogroup W135 of yen methoxy ninja ET discharge capsular saccharides (i) and tetanus toxoid (ii). PO copolymers, and (e) of serogroup X yen methoxy Ninja ET discharge of the capsule-type saccharides (i) and CRM197 (ii). It comprises a Po-polymer of methoxy Ninja ET discharge of the capsule-type saccharides (i) and tetanus toxoid (ii), and as a carrier protein PO copolymer containing tetanus toxoid is to enhance the immunogenicity of the included polymer containing CRM197 as the carrier protein . ≪ / RTI >

Composition IV is (a) Serum Group A ; PO copolymers, (b) a serogroup C yen methoxy ninja ET discharge capsular saccharides (i) and tetanus toxoid (ii). Four polymers of methoxy Ninja ET discharge of the capsule-type saccharides (i) and CRM197 (ii), (c) serogroup Y yen. PO copolymers, (d) serogroup W135 of yen methoxy Ninja ET discharge of the capsule-type saccharides (i) and CRM197 (ii). PO copolymers, and (e) of serogroup X yen methoxy Ninja ET discharge of the capsule-type saccharides (i) and CRM197 (ii). And a complex of a capsular saccharide (i) and a tetanus toxoid (ii) of meninitidis.

Composition V is (a) Serum Group A ; PO copolymers, (b) a serogroup C yen methoxy Ninja ET discharge of the capsule-type saccharides (i) and CRM197 (ii). Four polymers of methoxy Ninja ET discharge of the capsule-type saccharides (i) and CRM197 (ii), (c) serogroup Y yen. PO copolymers, (d) serogroup W135 of yen methoxy Ninja ET discharge of the capsule-type saccharides (i) and CRM197 (ii). PO copolymers, and (e) of serogroup X yen methoxy Ninja ET discharge of the capsule-type saccharides (i) and CRM197 (ii). And a complex of a capsular saccharide (i) and a tetanus toxoid (ii) of meninitidis.

Thus, in a first embodiment, the composition may comprise the serogroups A, C, Y, W135 and X saccharides in amounts of 0.5 μg to 10 μg, 0.5 μg to 5 μg, or 0.5 μg to 2 μg per 0.5 ml dose.

In another aspect of the first embodiment, the composition may comprise 10 μg of serogroup A sugars, 5 μg of serogroup C saccharides, 5 μg of serogroup W135 sugars, 5 μg of serogroup Y saccharides and 5 μg of serogroup X sugars.

Alternatively, the multivalent immunogenic composition may comprise 5 μg of serogroup A sugars, 5 μg of serogroup C saccharides, 5 μg of serogroup W135 sugars, 5 μg of serogroup Y saccharides and 5 μg of serogroup X sugars.

Therefore, in the second embodiment, one or more yen. The meninitidis saccharide formulations may be arbitrarily adsorbed to aluminum hydroxide, aluminum phosphate or a mixture of both, or may not be adsorbed arbitrarily to the adjuvant.

In one aspect of the second embodiment, the composition is capable of adding aluminum salt adjuvant in an amount of 20 μg to 300 μg, 20 μg to 200 μg, or 25 μg to 150 μg of Al ⁺⁺⁺ per 0.5 ml dose.

Another aspect of the second embodiment is that the aluminum salt adjuvant can be added in an amount of 25 μg to 125 μg of Al ⁺⁺⁺ per 0.5 ml.

A third embodiment of the present invention is that the composition may comprise a preservative selected from thiomucose and 2-phenoxyethanol.

One aspect of the third embodiment is that the composition may further comprise sodium phosphate, sodium chloride, or a combination thereof.

A fourth embodiment of the present invention is that the above-described polymorphic composition may be in a buffered liquid or lyophilized form.

One aspect of the fourth aspect is that the lyophilized immunogenic composition comprises: a) from 2% (w / v) to 5% (w / v) trehalose and from 0.25% to 0.75% sodium citrate; b) 2% (w / v) to 5% (w / v) sucrose and 0.25% to 0.75% sodium citrate; c) 2% (w / v) to 5% (w / v) sucrose, 2% (w / v) to 5% (w / v) lactose and 0.25% to 0.75% sodium citrate; And d) a stabilizer selected from 2% (w / v) to 5% (w / v) trehalose, 2% w / v lactose to 5% w / v lactose, and 0.25% ≪ / RTI >

Another aspect of the fourth embodiment is that the lyophilized immunogenic composition may further comprise a buffer selected from tris and phosphate.

Thus, in a fifth embodiment, the polysaccharides A, C, W, and X are mechanically sized to form between 100 kDa and 600 kDa, between 100 kDa and 400 kDa, preferably between 100 kDa and 200 kDa, And may have an average molecular weight between 100 kDa and 150 kDa. Mechanical sizing methods such as homogenization, microfluidization and high pressure cell disruption are preferred.

In another aspect of the fifth embodiment, the polysaccharide Y is chemically sized by a method selected from acid hydrolysis, alkaline degradation, oxidation with periodate, ozone decomposition, enzymatic hydrolysis, sonication, and electron beam fragmentation, 0.0 > kDa < / RTI > and 110 kDa. Preferably, the chemical sizing is carried out using sodium acetate at a temperature of 60 ° C to 80 ° C.

In the sixth embodiment, each of the circle. Meninitidis saccharides are incorporated into carrier proteins via a hetero or homo-bifunctional linker by cyanidation chemistry.

In one aspect of the sixth embodiment, the sized polysaccharide includes, but is not limited to, 1-cyano-4- (dimethylamino) -pyridinium tetrafluoroborate ('CDAP'), p-nitrophenyl cyanate And < / RTI > N-cyanotriethylammonium tetrafluoroborate (' CTEA '). In a preferred method of incorporation, the cyanide reagent is other than CDAP and is selected from the group consisting of 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT), 1-cyano-imidazole (1-CI) Benzotriazole (1-CBT), 2-cyanopyridazin-3 (2H) -one (2-CPO), or functional derivatives or modifications thereof .

In another aspect of the sixth embodiment, the activated polysaccharide or carrier protein, particularly the polysaccharide, is selected from the group consisting of hydrazine, a carbohydrazide, a hydrazine chloride, a dihydrazide or a mixture thereof and preferably an adipic acid dihydrate It reacts with Zaid.

Carbodiimide reaction, for example, in the presence of EDC, hydrazine, carbohydrazide, succinyl dihydrazide, adipic acid dihydrazide, hydrazine chloride (e.g., hydrazine dihydrochloride) Or any other dihydrazide, the hydrazide group can be introduced into the protein via the carboxyl group of the aspartic acid and the glutamic acid residue on the protein. EDC is used as a catalyst to activate and modify the protein reactants by hydrazine or dihydrazide. All water soluble carbodiimides, including EDC, can be used as catalysts. EDC-catalyzed proteins generally tend to polymerize and precipitate (Schneerson et al., Infect. Immun. 1986, 52: 519-528; Shafer et al., Vaccine 2000; 18 (13): 1273- 1281; and Inman et al., Biochemistry 1969; 8: 4074-4082).

B, C, H, I, K, L, 29E, W135, Y, and Z, which are individually incorporated into two or more different types of carrier proteins, in the seventh embodiment, wherein the polynuclear polysaccharide protein- ≪ / RTI > Capsule saccharides are selected from the meningococcal serogroups A, C, W135, Y and X, and the composition comprises sugars from one, two, three, four or five of these five serogroups. Certain compositions include serogroups A and X; Serogroups X and W135; Serogroups X and Y; Serum C and X; Serum groups A, Y, and X; Serogroups C, X and W135; Serum groups X, Y and W135; Serum groups A, C, and X; Serogroups Y, C, and X; Serum groups A, W, and X; Serogroups Y, W135, C, and X; Serogroups Y, W135, A and X; Serum group C, W135, A and X; Serogroups Y, C, A, and X; Serogroups A, C, Y, W135 and X. A composition comprising at least the serogroup X is preferred, and a composition comprising saccharides from all five serogroups is most preferred.

In aspects of the seventh embodiment, the carrier protein, but not limited to CRM197, diphtheria toxoid, tetanus toxoid, pertussis toxoid, a. Escherichia coli (E. coli) LT, a. E. coli ST, Pseudomonas rugi Ah Labor (Pseudomonas endoprotein A, outer membrane complex c (OMPC), porin, transferrin binding protein, neomolysin, pneumococcal surface protein A (PspA), hunting yeast attachment protein (PsaA), pneumococcal surface protein BVH-3 BVH-11, Bacillus Anthranilic cis protective antigen (PA), of Bacillus (Bacillus anthracis) Anthranilic cis detoxified with edema factor (EF) and lethal factor (LF), ovalbumin, keyhole of-rimpet H. linen (KLH), human serum albumin, bovine serum albumin (BSA) and a protein derivative purification tuberculin not (PPD ). ≪ / RTI > Preferably, the combination of carrier proteins used comprises tetanus toxoid and diphtheria toxoid, and CRM197 and tetanus toxoid.

In another aspect of the third embodiment, the inclusion reaction is carried out in the presence of adipic acid dihydrazide,? -Aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone, cystamine and p-nitrophenylethylamine Lt; / RTI > linker is used.

After incorporation, the conjugate can be purified from unreacted proteins and polysaccharides by any standard technique, including, in particular, size exclusion chromatography, density gradient centrifugation, ultrafiltration, hydrophobic interaction chromatography or ammonium sulfate fractionation (1986) J. Immunol. 137: 1181-1186; and HJ Jennings and C. Lugowski, (1981) J. Immunol. 127: 1011-1018).

In an eighth embodiment, the immunogenic compositions of the invention may further comprise additional non-meningococcal polysaccharide protein conjugates, such polysaccharides and oligosaccharides for use include, but are not limited to, serogroups 1, 2, 3 , Pneumococcal polysaccharides of 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F and 33F; To a brush loose influenza (Haemophilus influenzae) type b polysaccharide poly Lee view ribitol phosphate; Group B streptococcal polysaccharides of serotype III and V; Salmonella and blood ties (Salmonella typhi Vi polysaccharide. Other strains of pneumococcal and Group B streptococcal serotypes are also suitable for use herein and include other T-independent polysaccharides and oligosaccharide antigens such as Group A streptococci, Staphylococci, Enterococci, Klebsiella Pneumoniae (Klebsiella pneumoniae ) . Cola , Pseudomonas aeruginosa and Bacillus Polysaccharides or oligosaccharides derived from anthracis are also suitable. Bacterial polysaccharides and oligosaccharides are particularly preferred, but ginger bacterial lipopolysaccharides and lipid oligosaccharides, polysaccharides and oligosaccharide derivatives thereof, and viral polysaccharides and oligosaccharides are also used.

The compositions of the present invention can be provided and presented in a variety of ways. The composition may be provided as a vial, or the composition may be provided in a pre-filled syringe. The syringe can be supplied with or without needles. The syringe comprises a single dose of the composition, while the vial may comprise a single dose or multiple doses. The injectable composition is generally a liquid solution or suspension. Alternatively, the composition may be in solid form (e. G., Lyophilized) for a solution or suspension in a liquid vehicle prior to injection.

Example

Example  One:

Meningococcus  Preparation of polysaccharide X

a) fermentation and Meningococcus  X Purification of polysaccharides

And under optimum conditions in the fermentor using a suitable fermentation medium for a continuous fermentation system oil (continuous fed-batch fermentation mode) , yen. From methoxy Ninja ET display isolates (8210 and 9601) to obtain the X meningococcal polysaccharide. In addition, the meningococcal X capsular polysaccharide is typically prepared by a method comprising CTAB-based precipitation, ethanol (96%) treatment followed by deep filtration, carbon filtration, CaCl ₂ precipitation, ethanol (96%) treatment and ultrafiltration .

b) Meningococcus  Sizing of polysaccharides X

Purified meningococcal X polysaccharide is subjected to one to two passes of mechanical sizing (a constant system cell crusher) in WFI under a pressure of about 30 kpsi to 40 kpsi.

Example  2:

carrier  For protein Meningococcus  X polysaccharide Combination

a) Hydrazine The derivatized  tetanus Toxoid  ( TT )on Inclusive  Of various average molecular weights Meningococcus  X polysaccharide

First, 45 mg of homogenized X (strain 9601) polysaccharide (30 mg / ml) with an average molecular weight of 215 kD on SEC HPLC was activated with 90 mg of CDAP (dissolved in 100 mg / ml in acetonitrile) and the pH of the mixture And adjusted to 9.5 with 1 M NaOH. Then, after 3 minutes, 67.5 mg of hydrazine activated TT (30 mg / ml in 1 M NaCl) was added to the reaction. The reaction was monitored over HPLC and continued for up to 18 hours. After 18 hours, the reaction was quenched by the addition of glycine and the complex was purified by diafiltration of a 300 kD TFF membrane in 10 mM Tris, pH 7.2. Shodex columns SB-804 HQ and SB-805 HQ were sequentially used at a flow rate of 1 ml / min using PBS as the mobile phase. The polysaccharide concentration and protein concentration were determined by the phosphorous analysis method and the modified Rose analysis method, respectively.

Second, 60 mg of X (strain 8210) polysaccharide (24 mg / ml in 2 M NaCl), with an average molecular weight of 326 kD on SEC HPLC, was activated with 150 mg of CPPT (dissolved at 114 mg / ml in acetonitrile) Was adjusted to 9.5 with 2.5 M NaOH. Then, after 3 minutes, 37.5 mg of ADH-activated TT (37 mg / ml in 2 M NaCl) was added to the reaction. The reaction was monitored by HPLC and continued for up to 5 hours. After 5 hours, the reaction was stopped by the addition of glycine and the complex was purified by diafiltration of a 500 kD TFF membrane in 10 mM PBS, followed by 10 mM Tris, pH 7.2.

200 mg of X (strain 8210) polysaccharide (20 mg / ml in 1 M NaCl) with an average molecular weight of 120 kD on SEC HPLC was activated with 400 mg of CPIP (dissolved at 114 mg / ml in acetonitrile) and the pH of the mixture Was adjusted to 9.5 with 1 M NaOH. Then, after 3 minutes, 150 mg of ADH-activated TT (30 mg / ml) was added to the reaction. The reaction was monitored over HPLC and continued for up to 4 hours. After 4 hours, the reaction was stopped by the addition of glycine, and the complex was purified by diafiltration of a 300 kD TFF membrane in 10 mM PBS, followed by 10 mM Tris, pH 7.2.

b) ADH Activated < / RTI > and purified < RTI ID = Toxoid  ( TT )on Inclusive  Of various molecular weights Meningococcus  X polysaccharide

First, 200 mg of polysaccharide (27 mg / ml in 2M NaCl) of X (strain 8210) with an average molecular weight of 510 kD on SEC HPLC was activated with 400 mg of CPPT (dissolved in 114 mg / ml in acetonitrile) The pH was adjusted to 9.5 with 2.5 M NaOH. Then, after 3 minutes, 1.5 g of ADH (100 mg / ml in carbonate buffer) was added and the reaction was continued for up to 4 hours. After 4 hours, glycine was added and the reaction mixture was diafiltrated on an 8 kD TFF membrane. In addition, the ADH-Men X polysaccharide was concentrated. Purified TT (37.5 mg / ml in 0.9% NaCl) and MES pH 6.0 buffer were added to 44 mg (7.5 mg / ml) of this polysaccharide to make the final buffer strength of MES 100 mM, followed by 37.5 mg EDAC Dissolved, pH 6.0) was added. The reaction was continued for 4 hours and monitored on HPLC. Unbound polysaccharides were removed by gel filtration chromatography using a Toyopearl HW65 resin on an Akta chromatography system (GE Amersham). The fractions were collected and collected based on peak profile and saccharide-protein ratio.

Second, the polysaccharide of X (strain 8210), having an average molecular weight of 250 kD on SEC HPLC, was concentrated to 18 mg / ml. An amount of 200 mg was activated with 296 mg of CPPT (dissolved at 114 mg / ml in acetonitrile) and the pH of the mixture was adjusted to 9.5 with 2.5 M NaOH. Then, after 3 minutes, 1.12 g of ADH (100 mg / ml in carbonate buffer) was added and the reaction was continued for up to 4 hours. After 4 hours, glycine was added and the reaction mixture was diafiltrated on an 8 kD TFF membrane. The ADH-Men X polysaccharide was then concentrated. In addition, purified TT (36.7 mg / ml in 0.9% NaCl) and MES pH 6.0 buffer were added to 200 mg (7.5 mg / ml) of this polysaccharide and the final buffer strength of MES was adjusted to 100 mM, followed by 200 mg of EDAC (100 mM MES , PH 6.0) was added. The reaction was continued for 4 hours and monitored on HPLC. The complex was purified by diafiltration of a 500 kD TFF membrane in 10 mM PBS, followed by 10 mM Tris, pH 7.2.

According to the above data, final product yields of about 20% to 30% are achieved by i) a meningococcal X polysaccharide having an average molecular weight of about 100 kDa to 200 kDa, ii) an ADH-activated meningococcal X polysaccharide, iii) iv) the ratio of saccharide to protein between 0.5 and 2 during the conjugation reaction, v) CPPT as cyanation reagent, vi) incubation treatment at 2 ° C to 8 ° C, and vi) between 0.2 and 0.6 Lt; RTI ID = 0.0 > of the < / RTI > sugars to the protein.

Example  3:

carrier  protein CRM197 For Meningococcus  A, C, Y and W135  Polysaccharide Combination

Purified meningococcal polysaccharides A, C, Y, and W135 having an average molecular weight between 100 and 200 using a suitable cyanide reagent (CDAP or CPPT) were incorporated into CRM197 at a saccharide flux ratio of less than 1 protein. The complex was further purified by diafiltration on 300 kD TFF with 10 volumes of 10 mM PBS and 10 volumes of 10 mM Tris.

Example  4:

Two different carrier  Protein-containing Men  A, C, Y, W135  And X Composite Freeze-drying and formulation

Trehalose, sucrose and lactose . A lyophilized formulation was prepared containing a meninitartis discoide , sodium citrate and Tris buffer, wherein the free polysaccharide content was in the range and the moisture content was less than 2%. The stabilizer combination comprises a) 2% (w / v) to 5% (w / v) trehalose and 0.25% to 0.75% sodium citrate; b) 2% (w / v) to 5% (w / v) sucrose and 0.25% to 0.75% sodium citrate; c) 2% (w / v) to 5% (w / v) sucrose, 2% (w / v) to 5% (w / v) lactose and 0.25% to 0.75% sodium citrate; And d) from 2% (w / v) to 5% (w / v) trehalose, 2% w / v lactose to 5% w / v lactose and 0.25% to 0.75% sodium citrate .

Example  5:

Men  X sugar Composite  Containing Meningococcus  1 and 2 Composite  Biological activity of the composition

Six female Swiss albino mice weighing 16 g to 20 g were immunized with each formulation. Mice were immunized subcutaneously at days 0, 14, and 28. Each mouse was secondly immunized after 1 and 2 weeks and then collected (21 days and 35 days).

Antibody titration was performed by bead-based assay and SBA. Preimmunized serum samples from all 6 mice were mixed to prepare a single mixed serum for each formulation from Study 4 and also from each of the 6 Albino strains belonging to the Swiss albino strain for each formulation Immunization serum samples were mixed and pools 1, 2 and 3 were prepared using serum from mouse 1 + 2, mouse 3 + 4 and mouse 5 + 6, respectively. Each pool was analyzed for total IgG titers (multiple bead-based assay) and functional antibody titers (SBA).

The mouse immunogenicity data indicates that liquid and lyophilized compositions of polyhydric capsules containing monovalent X-tetanus toxoid conjugate and X-tetanus toxoid conjugate were found to be immunogenic. In addition, X- tetanus toxoid PO copolymer 1μg, 1 is a liquid composition containing the sodium chloride, thio meosal and Al ⁺⁺⁺ 125μg provides the best immunogenic reaction. In addition, a liquid polyvalent composition 0.5 containing 1 μg of each of the A-CRM197, C-CRM197, Y-tetanus toxoid, W-CRM197 and X-tetanus toxoid conjugate and 5 sugars of each, sodium chloride, thiomercury and Al ⁺⁺⁺ ml provides the optimal immunogenic response. Therefore, in such a pentapeptide composition, a collagen containing tetanus toxoid as a carrier protein has been found to enhance the immunogenicity of a CRM197-containing collagen as a carrier protein.

It is to be understood that the illustrated embodiments are merely preferred embodiments of the invention and are not intended to limit the scope of the invention in view of the many possible embodiments to which the principles of the invention disclosed may be applied. Rather, the scope of the invention is defined by the following claims. Accordingly, the inventors contemplate all that fall within the scope and spirit of these claims.

Claims (37)

yen. Methoxy Ninja ET display (N. meningitidis) polysaccharides X - as an immunogenic composition comprising the protein PO copolymer (conjugate),
The encapsulated saccharide is selected from the group consisting of : Wherein the immunogenic composition is derived from a strain of Meninitidis . The method according to claim 1, further one or more of polysaccharide polymer PO is a yen derived from serogroup A, B, C, W135 and Y. Wherein the immunogenic composition comprises a meningitidis capsular saccharide. The method according to claim 1, wherein the yen. A methoxy Ninja ET discharge X strain is selected from 8210, 9601, 9592, 9554 and 2526, the immunogenic composition. The composition of claim 2, wherein said composition comprises two or more different carrier proteins to conjugate all five polysaccharides . A methoxy Ninja ET discharge serogroup A, comprises each of the capsule polysaccharide of C, W135, Y, and X, the immunogenic composition. The method according to claim 1 or 4, wherein each said yen. A methoxy Ninja ET discharge saccharides is conjugated to a carrier protein selected from the group consisting of who TT, DT, CRM197, fragment C of TT, protein D, OMPC and New Mora, immunogenic compositions. The method of claim 5, wherein each said yen. A methoxy Ninja ET discharge saccharides is conjugated to a carrier protein selected from the group consisting of TT, DT CRM197 and, immunogenic compositions. 7. The method of claim 6, wherein each said yen. Wherein the meninitidis saccharide is conjugated to a carrier protein selected from the group consisting of TT and DT. The method according to claim 4, wherein the composition comprises (a) serogroup A yen. PO copolymers, (b) a serogroup C yen methoxy ninja ET discharge capsular saccharides (i) and tetanus toxoid (ii). A mixture of capsular saccharide (i) and tetanus toxoid (ii) of meninitidis, (c) serum group Y yen. PO copolymers, (d) serogroup W135 of yen methoxy ninja ET discharge capsular saccharides (i) and diphtheria toxoid (ii). PO copolymers, and (e) of serogroup X yen methoxy ninja ET discharge capsular saccharides (i) and tetanus toxoid (ii). The capsular saccharides (i) of methoxy Ninja ET display and comprises a Po-polymer of tetanus toxoid (ii), the immunogenic composition. The method according to claim 4, wherein the composition comprises (a) serogroup A yen. PO copolymers, (b) a serogroup C yen methoxy Ninja ET discharge of the capsule-type saccharides (i) and CRM197 (ii). Four polymers of methoxy Ninja ET discharge of the capsule-type saccharides (i) and CRM197 (ii), (c) serogroup Y yen. PO copolymers, (d) serogroup W135 of yen methoxy ninja ET discharge capsular saccharides (i) and tetanus toxoid (ii). PO copolymers, and (e) of serogroup X yen menin chair ET discharge capsular saccharides (i) and CRM197 (ii). It comprises a Po-polymer of methoxy Ninja ET discharge of the capsule-type saccharides (i) and tetanus toxoid (ii), and as a carrier protein PO copolymer containing tetanus toxoid is to enhance the immunogenicity of the included polymer containing CRM197 as the carrier protein ≪ RTI ID = 0.0 > 1, < / RTI > The method according to claim 4, wherein the composition comprises (a) serogroup A yen. PO copolymers, (b) a serogroup C yen methoxy ninja ET discharge capsular saccharides (i) and tetanus toxoid (ii). Four-polymer, (c) serogroup Y yen methoxy ninjayi of discharge tee capsular saccharides (i) and CRM197 (ii). PO copolymers, (d) serogroup W135 of yen methoxy ninja ET discharge capsular saccharides (i) and tetanus toxoid (ii). PO copolymers, and (e) of serogroup X yen methoxy Ninja ET discharge of the capsule-type saccharides (i) and CRM197 (ii). A methoxy Ninja ET discharge of the capsule-type saccharide comprises a Po-polymer of (i) and CRM197 (ii), the immunogenic composition. The method according to claim 4, wherein the composition comprises (a) serogroup A yen. PO copolymers, (b) a serogroup C yen methoxy ninja ET discharge capsular saccharides (i) and tetanus toxoid (ii). Four polymers of methoxy Ninja ET discharge of the capsule-type saccharides (i) and CRM197 (ii), (c) serogroup Y yen. PO copolymers, (d) serogroup W135 of yen methoxy Ninja ET discharge of the capsule-type saccharides (i) and CRM197 (ii). PO copolymers, and (e) of serogroup X yen methoxy Ninja ET discharge of the capsule-type saccharides (i) and CRM197 (ii). The capsular saccharides (i) of methoxy Ninja ET display and comprises a Po-polymer of tetanus toxoid (ii), the immunogenic composition. The method according to claim 4, wherein the composition comprises (a) serogroup A yen. PO copolymers, (b) a serogroup C yen methoxy Ninja ET discharge of the capsule-type saccharides (i) and CRM197 (ii). Four polymers of methoxy Ninja ET discharge of the capsule-type saccharides (i) and CRM197 (ii), (c) serogroup Y yen. PO copolymers, (d) serogroup W135 of yen methoxy Ninja ET discharge of the capsule-type saccharides (i) and CRM197 (ii). PO copolymers, and (e) of serogroup X yen methoxy Ninja ET discharge of the capsule-type saccharides (i) and CRM197 (ii). The capsular saccharides (i) of methoxy Ninja ET display and comprises a Po-polymer of tetanus toxoid (ii), the immunogenic composition. 5. The immunogenic composition of claim 4, wherein each of said capsular polysaccharides has an average size between 100 kDa and 600 kDa. 14. The immunogenic composition of claim 13, wherein each of said capsular polysaccharides has an average size between 100 kDa and 300 kDa. 15. The immunogenic composition of claim 14, wherein each of said capsular polysaccharides has an average size between 100 kDa and 200 kDa. 5. The method of claim 4, wherein the serogroups A, C, W135, and three or more yen from X-post-sizing . Wherein the meninitidis polysaccharide has an average size between 100 kDa and 150 kDa. 17. The immunogenic composition of claim 16, wherein said post-sizing is performed using a high pressure cell disruption system. 5. The method of claim 4, wherein the antioxidant from the serogroup Y post-chemical sizing . Wherein the meninitidis polysaccharide has an average size between 90 kDa and 110 kDa. 19. The immunogenic composition of claim 18, wherein said post-chemical sizing is performed using sodium acetate at a temperature of from 60 DEG C to 80 DEG C. 5. The method of claim 4, wherein each said yen. Methoxy Ninja ET discharge saccharides homo or hetero cyanide by conjugation chemistry-would in through the bifunctional linker (homo or hetero-bifunctional linker) is conjugated to the carrier protein, immunogenic compositions. 21. The immunogenic composition of claim 20, wherein the linker is ADH. The method of any one of claims 1 to 21, wherein the cyanide reagent is selected from the group consisting of 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT), 1-cyano- Selected from the group consisting of cyanobenzotriazole (1-CBT), 2-cyanopyridazin-3 (2H) -one (2-CPO) or a functional derivative or modification thereof ≪ / RTI > The method according to claim 20, wherein the yen. The meninitidis X-saccharide-tetanus toxoid conjugate comprises i) polysaccharide sizing, ii) CPPT-based activation of a sized polysaccharide having an average molecular weight between 100 kDa and 150 kDa at a pH between 9 and 9.5, iii) Followed by ADH addition for a period of 3 to 3 minutes followed by incubation for 4 to 20 hours, iv) unreacted ADH removal by diafiltration, and v) ADH activation at a ratio between 0.75 and 1.5 in the presence of MES buffer and EDAC Wherein the complexing reaction is carried out at a temperature between 2 ° C and 8 ° C and the protein is reacted with the protein in the final complex, Wherein the ratio of saccharides is between 0.2 and 0.6. The method according to claim 20, wherein the yen. The meninitidis X-saccharide-tetanus toxoid conjugate is characterized by i) polysaccharide sizing, ii) CPPT-based activation of the sized polysaccharide with an average molecular weight between 100 kDa and 150 kDa at a pH between 9 and 9.5, iii) After 3 minutes, by addition of an ADH-activated carrier protein at a ratio of saccharides to between 0.5 and 2, followed by incubation for 2 to 20 hours, wherein the incorporation reaction is carried out at a temperature of about < RTI ID = Wherein the ratio of the saccharide to the protein in the final formulation is between 0.2 and 0.6. The composition according to claim 1 or 2, wherein the composition comprises the serogroups A, C, Y, W135 and X saccharides in an amount of 0.5 μg to 10 μg, 0.5 μg to 5 μg, or 0.5 μg to 2 μg per 0.5 ml dose. Immunogenic composition. 26. The immunogenic composition of claim 25, wherein said composition comprises 10 μg of serogroup A sugars, 5 μg of serogroup C saccharides, 5 μg of serogroup W135 sugars, 5 μg of serogroup Y saccharides and 5 μg of serogroup X sugars. 26. The immunogenic composition of claim 25, wherein said composition comprises 5 mu g of serogroup A sugars, 5 mu g of serogroup C saccharides, 5 mu g of serogroup W135 saccharides, 5 mu g of serogroup Y sugars and 5 mu g of serogroup X sugars. 26. The method of claim 25, wherein one or more of the yen. Wherein the meninitidis saccharide conjugate is optionally adsorbed to aluminum hydroxide, aluminum phosphate or a mixture of both, or is not adsorbed arbitrarily to the adjuvant. 29. The immunogenic composition of claim 28, wherein said composition comprises the step of adding an aluminum salt adjuvant in an amount of 20 μg to 300 μg, 20 μg to 200 μg, or 25 μg to 150 μg of Al ⁺⁺⁺ per 0.5 ml dose. 29. The immunogenic composition of claim 29, wherein said composition comprises the step of adding an aluminum salt adjuvant in an amount of from 25 μg to 125 μg of Al ⁺⁺⁺ per 0.5 ml. 30. The immunogenic composition of claim 28, wherein said composition further comprises a preservative selected from thiomarch and 2-phenoxyethanol. 32. The immunogenic composition of claim 31, wherein said composition comprises sodium phosphate, sodium chloride, or a combination thereof. 32. The immunogenic composition according to any one of claims 1 to 32, wherein the sugar form is in a buffered liquid form. 34. The immunogenic composition of any one of claims 1 to 33, wherein the sugar capsule is in a lyophilized form. 34. The lyophilized immunogenic composition of claim 34,
The stabilizer combination comprises a) 2% (w / v) to 5% (w / v) trehalose and 0.25% to 0.75% sodium citrate; b) 2% (w / v) to 5% (w / v) sucrose and 0.25% to 0.75% sodium citrate; c) 2% (w / v) to 5% (w / v) sucrose, 2% (w / v) to 5% (w / v) lactose and 0.25% to 0.75% sodium citrate; And d) from 2% (w / v) to 5% (w / v) trehalose, 2% w / v lactose to 5% w / v lactose and 0.25% to 0.75% sodium citrate Lt; RTI ID = 0.0 > immunogenic < / RTI > composition. 36. The lyophilized immunogenic composition of claim 35, wherein said composition further comprises a buffer selected from tris and phosphate. Nyseria Eighty-mail Ninja display (Neisseria As a method of immunizing a human host against a disease caused by infection with meningitidis ,
Wherein the immunization method comprises administering to the host an immunogenic composition or vaccine of any one of claims 1 to 36 in an immunoprotective amount.

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